EP3595702B1

EP3595702B1 - Modified haemoglobin proteins

Info

Publication number: EP3595702B1
Application number: EP18711670.2A
Authority: EP
Inventors: Chris E. Cooper; Brandon REEDER; Gary SILKSTONE
Original assignee: University of Essex Enterprises Ltd
Current assignee: University of Essex Enterprises Ltd
Priority date: 2017-03-13
Filing date: 2018-03-13
Publication date: 2023-04-26
Anticipated expiration: 2038-03-13
Also published as: EP3595702A1; US20200131247A1; GB201704006D0; WO2018167469A1; ZA201905544B

Description

Field of the Invention

Certain aspects of the present invention relate to a modified human haemoglobin protein comprising at least one modified human haemoglobin chain subunit, with enhanced, in comparison to a reference protein, reduction of an iron ion associated with the modified protein. Also included herein are such modified human haemoglobin proteins and compositions comprising such modified human haemoglobin proteins for use as a medicament e.g. in the treatment of ischemia and/or hypoxia.

Background to the Invention

In cases where major blood loss occurs or blood flow is reduced, such as in cases of where blood flow is reduced due to blood loss due to trauma, disease or ischemia, tissue damage and organ dysfunction can occur due to oxygen deprivation of the cells and tissue leading to hypoxia, anoxia and in some cases cell death.
The use of blood transfusions (red blood cell transfusion) can help transport oxygenated blood through the major blood vessels but not always to smaller capillaries and the microvasculature meaning that these may remain collapsed and in an ischemic condition even after treatment with expanders, drugs or by a blood transfusion.
There also exist a number of issues with red blood cell transfusions. In some cases, blood for use in a red blood cell transfusion may not be readily available such as on the battlefield, in pre-hospital emergency treatment and during major civil crises involving mass casualties. The shelf-life of donated blood and its stringent handling needs also mean that suitable donated blood may not be readily available for transfusion. Issues also arise where patients have rare blood types that are not easily matched or may not accept blood transfusions for religious or personal reasons.
One solution to these problems is the use of a blood substitute. A blood substitute is an oxygen carrying solution that can help to maintain the oncotic pressure needed to maintain blood volume and transport oxygen to cells and tissue around the body therefore helping prevent oxygen deprivation (hypoxia) and ischemia.
Blood substitutes can act as an oxygenation bridge till red blood cell transfusion or in some cases used instead of red blood cell transfusion. It is also possible to use the oxygen carrying constituent of a blood substitute as an oxygen therapeutic. An oxygen therapeutic can be used to help improve the oxygen carrying ability of a patient's blood as well as improving the oxygen carrying capabilities of blood used for transfusions when administered in addition to red blood cells. Oxygen therapeutics as part of a blood substitute or as part of other fluids can be used in a number of methods wherein oxygen may be required such as in the storage of organs as well as in the treatment of carbon monoxide poisoning and in cell culture methods as well.
There are currently two main types of oxygen carrying therapeutics undergoing studies, fluorocarbon emulsions and haemoglobin based oxygen carriers (HBOCs).
Adult haemoglobin in its native environment of a red blood cell is a tetrameric protein composed of two alpha and two beta globin chain subunits, each subunit carrying a haem molecule. One alpha-like globin chain and one beta-like globin chain combine to form a stable dimer. The two dimers are then aligned in an anti-parallel fashion to form a tetramer. The binding between dimers in the tetramer is not as strong as monomers binding to form dimers. Therefore tetramers have a tendency to dissociate back to dimers. At high globin concentrations the tetramer form is the most common but when diluted dimers are the most predominant form.
A disadvantage associated with the use of native haemoglobin as an oxygen therapeutic is that the tetrameric form readily dissociates into the dimeric form which is rapidly cleared by the kidneys causing damage to the kidneys and renal system.
In order to address this disadvantage, a number of haemoglobin variants which prevent dissociation of the tetramer and that are more stable have been designed. Another approach has been to modify the protein by the addition of polymers to improve stability.
Another disadvantage of HBOCs has been spontaneous oxidation of the haem iron atom under physiological conditions. The haem iron atom is converted from the active oxygen carrying ferrous (Fe²⁺) oxy-haem form to the non-functional (non-oxygen carrying) ferric (Fe³⁺) met-haem form. This oxidation can also produce a superoxide ion (O₂·^-) which subsequently dismutates rapidly to H₂O₂. The H₂O₂ if not degraded by catalases can react with the Fe³⁺ ion to produce a highly reactive and cytotoxic ferryl (Fe⁴⁺) haem form and protein based free radicals. This oxidative cascade can be damaging as H₂O₂ is a powerful oxidant known to produce cellular damage and the ferryl haem and protein based free radicals can initiate oxidation of lipids, nucleic acids and amino acids within the cell. The free radicals produced can also lead to damage to the protein and the haem group, which can cause the release of one or more haem groups which can have a number of adverse effects on cells such as causing inflammation.
It has been previously shown that it is possible to increase reduction of the ferryl haem form to the non-functional ferric met-haem form by the introduction of redox-active amino acid residues at specific locations into the protein therefore reducing toxicity of HBOCs. The mutations are believed to introduce an electron transport pathway not previously present in certain haemoglobin chain subunits or certain other haemoglobin like proteins. It was found though that these mutations did not lead to further reduction of the non-functional ferric met-haem form to the functional ferrous oxy-haem form. Therefore, these mutated HBOCs displayed lower toxicity but no improved oxygen carrying capability.
XP055479136 (Alayash et al., Journal of Biological Chemistry, 22 January 1999, pages 2029-2037) and XP055479275 (Carver et al., Journal of Biological Chemistry, 15 July 1992, pages 14443-14450) disclose a L29F sperm whale myoglobin variant. DATABASE UniProt [Online] 15 February 2017 (XP055811938, retrieved from www.uniprot.org accession no. Q90486) discloses a zebrafish haemoglobin beta 1 subunit.
These disadvantages associated with prior HBOC oxygen therapeutics mean that there is a still a need for HBOCs with improved oxygen carrying capability as well as reduced toxicity.
It is an aim of certain embodiments of the present invention to at least partly mitigate the above-mentioned problems associated with the prior art.

Summary of the Invention

In a first aspect of the present invention there is provided a modified human haemoglobin protein comprising at least one modified human haemoglobin chain subunit as defined in any one of claims 1 to 5.
In a second aspect of the present invention there is provided a composition comprising;

a modified human haemoglobin chain protein comprising at least one modified human haemoglobin chain subunit as defined in any one of claims 1 to 5; and
a pharmaceutically acceptable carrier or diluent.

In certain embodiments, the composition further comprises at least one reductant. In certain embodiments, the at least one reductant is ascorbate.
In certain embodiments, the composition is a pharmaceutical composition. In certain embodiments, the composition is a blood substitute composition.
In another aspect of the present invention there is provided a composition as defined in any one of claims 6 to 9 for use as a medicament.
In certain embodiments, the composition is for use in the treatment of ischemia. In certain embodiments, the composition is for use in the treatment of hypoxia. In certain embodiments, the composition is for use in the treatment of ischemia and/or hypoxia.
Further details of certain embodiments are provided below.

Brief Description of Drawings

Certain embodiments of the present invention will now be described hereinafter, by way of example only, with reference to the accompanying drawings in which:

Figure 1 illustrates the rate of autoxidation of 10 µM of recombinant adult haemoglobin (HbA) ferrous oxy-haemoglobin to ferric met-haemoglobin for various reference proteins (wild-type HbA (wt) and V1M modified wild-type HbA (wt V1M)), the βT84Y modified protein and βT84Y (V1M) modified protein in sodium phosphate buffer (20 mM, pH 7.2) at a temperature of 37ºC. Each bar is an average of 6 repeat experiments. The standard deviations are shown by the error bars. Rate traces were fitted to single exponentials;
Figure 2 illustrates the rate of haem loss from 1.7 µM recombinant HbA ferric met-haemoglobin for various reference proteins (wild-type HbA (wt) and V1M modified wild-type HbA (wt V1M)) and βT84Y modified protein and βT84Y (V1M) modified protein, to 2 µM hemopexin at 37ºC in sodium phosphate buffer (20 mM, pH 7.2). The bars are an average of 3 repeat experiments, standard deviations are shown by the error bars;
Figure 3 illustrates the percentage of ferric met-haemoglobin formed from ferryl haemoglobin (10 µM) after incubation with ascorbate (30 µM) at 25ºC in sodium phosphate buffer (20 mM, pH 7.2) by various modified recombinant HbA proteins including beta chain subunit modifications and wild-type recombinant HbA. Each bar represents an average of 3 repeat experiments, standard deviations are shown by the error bars;
Figure 4 illustrates the percentage, by various modified recombinant HbA proteins including at least one or more beta or alpha chain subunit modifications and wild-type recombinant HbA protein, of ferrous oxy-haemoglobin formed from ferric met-haemoglobin (10 µM) in the presence of ascorbate (100 µM) at 25ºC in sodium phosphate buffer (20 mM, pH 7.2).
Figure 5 illustrates a comparison of the capability of 4.4 µM of wild-type (wt) (long dashed line) and 4.4 µM βT84Y modified (short dashed line) recombinant HbA proteins to reduce ferric met-haemoglobin to ferrous oxy-haemoglobin over a time period of 360 minutes in the presence of ascorbate (100 µM) at 25ºC in sodium phosphate buffer (20 mM, pH 7.2). The black solid line shows the initial ferric met-haemoglobin spectrum;
Figure 6 illustrates the lack of a correlation between ferryl haemoglobin reduction ability and ferric met-haemoglobin reduction ability for various modified recombinant HbA proteins;
Figure 7 illustrates a comparison of the percentage of ferrous (oxy-haemoglobin) formation from ferric (met-haemoglobin) for various modified recombinant foetal haemoglobins (HbF) in comparison to reference proteins (wild type (WT) and αV1M, γG1M modified HbF). Time scale of measurements was 60 minutes. Buffer used was sodium phosphate (20 mM, pH 7.2) and experiments were carried out at a temperature of 25°C with haem present at 10 µM; and ascorbate present at 100 µM;
Figure 8 illustrates the amino acid sequences of:
- wild-type human haemoglobin beta chain subunit (SEQ. ID. NO. 1);
- wild-type human haemoglobin alpha chain subunit (SEQ. ID. NO. 2);
- wild-type human haemoglobin gamma 1 chain subunit (also known as gammaA) (SEQ. ID. NO. 3);
- wild-type human haemoglobin gamma 2 chain subunit (also known as gammaG) (SEQ. ID. NO. 4); and
- haemoglobin beta chain with the modification V1M (SEQ. ID. NO. 5);
Figure 9 illustrates the amino acid sequences of:
- haemoglobin alpha chain with the modification V1M (SEQ. ID. NO. 6);
- haemoglobin gamma 1 chain subunit with the modification G1M (SEQ. ID. NO. 7);
- haemoglobin gamma 2 chain subunit with the modification G1M (SEQ. ID. NO. 8);
- haemoglobin beta chain subunit with the modification T84Y (SEQ. ID. NO. 9); and
- haemoglobin beta chain subunit with modifications V1M and T84Y (SEQ. ID. NO.10);
Figure 10 illustrates the amino acid sequences of:
- haemoglobin alpha chain subunit with the modification V1M and L91Y (SEQ. ID. NO. 11);
- haemoglobin alpha chain subunit with the modification L91Y (SEQ. ID. NO. 12);
- haemoglobin gamma 2 chain subunit with modifications G1M and L96Y (SEQ. ID. NO. 13);
- haemoglobin alpha chain subunit with the modifications V1M and L29F (SEQ. ID. NO. 14); and
- haemoglobin gamma 2 chain subunit with the modifications G1M and V67F (SEQ. ID. NO. 15); and
Figure 11 illustrates the amino acid sequences of:
- haemoglobin alpha chain subunit with the modifications V1M, L29F and L91Y (SEQ. ID. NO. 16);
- haemoglobin gamma 2 chain subunit with the modifications G1M, V67F and L96Y (SEQ. ID. NO. 17); and human myoglobin (SEQ. ID. NO. 18).
Figure 12 illustrates a comparison of the rate of formation of ferrous (oxy-haemoglobin) formation from ferric (met-haemoglobin) for various modified recombinant foetal haemoglobins (HbF) in comparison to reference protein (wild type, WT). The time course (577 - 630 nm) was fitted to a single exponential function. The rate constants were plotted against ascorbate concentration and this was fitted to a straight line to determine the second order rate constant. Buffer used was sodium phosphate (70 mM, pH 7.2) and experiments were carried out at a temperature of 25°C with haem present at 10 µM. Significantly increased rates of reduction (ferric-met to ferrous) were seen compared to wild-type for the mutations αL91Y, γF85Y and γL96Y.
Figure 13 illustrates the low correlation between autoxidation (ferrous to ferric-met) and ascorbate reduction (ferric-met to ferrous) for recombinant HbA proteins.
Figure 14 illustrates the lack of a correlation between autoxidation (ferrous to ferric-met) and ascorbate reduction (ferric-met to ferrous) for recombinant HbF proteins.

Detailed Description of the Certain Embodiments

Further features of certain embodiments of the present invention are described below.
The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art.
Most general molecular biology, microbiology recombinant DNA technology and immunological techniques can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2001) Cold Harbor-Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al., Current protocols in molecular biology (1990) John Wiley and Sons, N.Y. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., Academic Press; and the Oxford University Press, provide a person skilled in the art with a general dictionary of many of the terms used in this disclosure.
Units, prefixes and symbols are denoted in their Système International de Unitese (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. All amino acid residues in proteins of embodiments of the invention are preferably of the L-configuration. However, D-configuration amino acids may also be present.
In a broad aspect, the present disclosure relates to a modified human haemoglobin protein comprising at least one modified human haemoglobin chain subunit as defined in claim 1 and medical uses thereof as defined in claims 10 and 11.
As used herein the term "oxygen-carrying protein" refers to any polypeptide chain that in its native state is able, alone or in complex with other molecules and/or polypeptides, to bind to oxygen, transport oxygen and subsequently release oxygen bound to the protein, therefore is a polypeptide that releasably binds to oxygen.
As used herein the terms "polypeptide" and "protein" are terms that are used interchangeably to refer to a polymer of amino acids, without regard to the length of the polymer. Typically, polypeptides and proteins have a polymer length that is greater than that of "peptides."
As used herein, the term "wild-type" refers to an amino acid sequence or nucleic acid sequence that is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that are found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modifications of the wild-type sequence).
In certain embodiments, the wild-type protein is a human haemoglobin beta chain subunit as set forth in SEQ. ID. No. 1. In certain embodiments, the wild-type protein is a human haemoglobin gamma 1 chain subunit (also known as gamma-A) as set forth in SEQ. ID. No. 3. In certain embodiments, the wild-type protein is a human haemoglobin gamma 2 chain subunit (also known as gamma-G) as set forth in SEQ. ID. No. 4.
The modified human haemoglobin protein exhibits enhanced reduction of a ferric (Fe³⁺) ion to a ferrous (Fe²⁺) ion as compared to the reference protein as defined in claim 1.
The kinetic and thermodynamic stability of the ferrous-oxy state can be measured by the spontaneous oxidation (autoxidation) of the ferrous-oxy state to the ferric-met state. However, this stability does not inform the ability of external reductants to re-convert that ferric-met state back to the functional ferrous-oxy state, which is largely a function of kinetic limitations that are not easy to predict a priori. Thus, it is to be understood herein that there is a difference between modifications that aim to stabilise the ferrous form (e.g. by preventing autoxidation) and those that aim to enhance reduction of the ferric form to the ferrous by external reductants. In other words, autoxidation does not predict, nor does it correlate with, reduction of ferric to ferrous ions. As shown herein, there is a lack of correlation between autoxidation (ferrous to ferric-met) and ascorbate reduction (ferric-met to ferrous) for both recombinant HbA proteins (Figure 13) and recombinant HbF proteins (Figure 14). Not only are the slopes of the correlation plot not significant, but they are positive. i.e. if anything those mutants with enhanced ferrous stability are, counter-intuitively, those with decreased ferric to ferrous reduction rates.
As such, the invention relates to the unexpected finding that the addition of the redox-active amino acid, tyrosine, at the positions of the modified human haemoglobin protein as defined in claim 1, may act to facilitate electron transfer and result in the rapid reduction of the at least one iron ion.
In certain embodiments, the "reference protein" is a wild-type version of the modified human haemoglobin protein. Alternatively, the reference protein may comprise one or more further modifications compared to the wild-type protein.
In certain embodiments, the reference protein may comprise a modification which substitutes the first (N-terminal) amino acid residue substituted with a methionine. For example, the reference protein may be a human haemoglobin beta chain subunit in which the first amino acid residue (valine) of a wild-type human haemoglobin beta chain subunit has been substituted with a methionine as set forth in SEQ. ID. No. 5 (also referred to as βV1M).
In certain embodiments, the reference protein may be a human haemoglobin gamma 1 chain subunit in which the first amino acid residue (glycine) of the wild-type human haemoglobin gamma 1 chain subunit has been substituted with a methionine as set forth in SEQ. ID. No. 7 (also referred to as γ1G1M).
In certain embodiments, the reference protein may be a human haemoglobin gamma 2 chain subunit in which the first amino acid residue (glycine) of the wild-type human haemoglobin gamma 2 chain subunit has been substituted with a methionine as set forth in SEQ. ID. No. 8 (also referred to as γ2G1M).
The modifications described above may help with recombinant expression, purification and/or isolation of a protein.
Throughout this specification, the conventional one letter and three letter codes for naturally occurring amino acids are used, as well as generally accepted three letter codes for other amino acids.
The term "associated with" as used herein refers to an interaction between two or more molecules wherein the molecules are bound together, indirectly bound together or partially bound to each other.
As used herein the terms "bound", "bind" and "bonding" may relate to any form of attractive interaction that may occur between two or more molecules. Non-limiting examples of binding are Van Der Waals interactions, Dipole to Dipole interactions, hydrophobic interactions, Hydrogen bonding, electrostatic bonding, covalent bonding, metallic bonding and ionic bonding.
In certain embodiments, the modified human haemoglobin protein is an isolated protein.
The term "isolated" as used herein refers to a polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the polypeptide is purified:

(1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or
(2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain.

Isolated polypeptides include polypeptides in situ within recombinant cells, since at least one component of the human haemoglobin polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptides will be prepared by at least one purification step.
The at least one modification as defined in claim 1 enhances the reduction of at least one ferric ion coordinated within at least one haem molecule to at least one ferrous (Fe²⁺) ion.
As used herein, the terms "haem" and "heme" refer to a type of porphyrin molecule wherein the metal ion coordinated within the central cavity of the heterocyclic ring is an iron ion. In certain embodiments, the iron ion is covalently coordinated.
In certain embodiments, the at least one haem is haem B. In certain embodiments, the haem is at least one or more of haem A, haem C, haem O, haem I, haem m, haem D and/or haem S. Other suitable naturally occurring and non-naturally occurring porphyrins and haems will be known to those skilled in the art.
Haemoglobins are tetrameric proteins made up of four polypeptide subunits each of which comprise a haem molecule. Haemoglobins constitute the oxygen carrying component of blood contained within red blood cells. As blood circulates through the lungs, the oxygen present in the alveolar capillaries diffuses through the alveolar membrane and acts to convert haemoglobin within the red blood cells to a reversible molecular complex known as oxy-haemoglobin. Because the association of the oxygen and haemoglobin is reversible, the oxygen molecules are gradually released from the haemoglobin when blood reaches the tissue capillaries. Eventually, the oxygen molecules diffuse into the tissues and is consumed by metabolism. As the oxygen is released, oxy-haemoglobin reduces to haemoglobin.

The modified haemoglobin is a human haemoglobin. In certain embodiments, the haemoglobin is a human adult haemoglobin. In certain embodiments, the haemoglobin is a human foetal haemoglobin. Non-limiting examples of naturally occurring human haemoglobins are given in Table 1. The most common form of haemoglobin found in humans is α₂β₂ (also referred to as adult haemoglobin) i.e. it is composed of two alpha chain subunits and two beta chain subunits. An example of foetal haemoglobin is α₂γ₂ (i.e. it is composed of two alpha chain subunits and two gamma chain subunits).

Table 1: Types of human haemoglobins

Name	Subunits
Gower 1 (not claimed)	ζ₂ε₂
Gower 2 (not claimed)	α₂ε₂
Haemoglobin Portland I	ζ₂γ1₂ or ζ₂γ2₂
Haemoglobin Portland II	ζ₂β₂
Haemoglobin F	α₂γ1₂ or α₂γ2₂
Haemoglobin A	α₂β₂
Haemoglobin A₂ (not claimed)	α₂δ₂
Haemoglobin H	β₄
Haemoglobin Barts	γ₄

In certain embodiments, the haemoglobins listed in Table 1 are reference proteins as referred to herein.
The modified human haemoglobin comprises at least one modified human haemoglobin chain subunit. In certain embodiments, the modified human haemoglobin protein is a modified human haemoglobin chain subunit.
In certain embodiments, the modified human haemoglobin protein is a human haemoglobin beta chain subunit. In certain embodiments, the modified human haemoglobin protein is a human haemoglobin gamma chain subunit. In certain embodiments, the modified human haemoglobin protein is a human haemoglobin gamma 1 chain subunit. In certain embodiments, the modified human haemoglobin protein is a human haemoglobin gamma 2 chain subunit.
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin beta chain subunit wherein the at least one modification is βT84Y. In certain embodiments, the at least one modification is βF85Y.
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin beta chain subunit wherein the at least one modification is a plurality of modifications selected from one or more of βT84Y and/or βF85Y.
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin gamma 1 chain subunit wherein the at least one modification is γ1L96Y.
In certain embodiment, the modified human haemoglobin protein comprises at least one human haemoglobin gamma 2 chain subunit wherein the at least one modification is γ2L96Y.
In certain embodiments, the modified human haemoglobin protein may comprise at least one further modification as compared to the reference protein. By way of example, the modified protein may comprise one, two three, four, five, six or more additional amino acid residue substitutions, deletions and/or insertions (which may be contiguous or non-contiguous). These further modifications may affect further properties of the modified protein such as oxygen affinity or cooperativity, stability and assembly rate, decreased porphyrin loss, decreased metallic ion autoxidation rate, resistance to proteolytic degradation, decreased aggregation, nitric oxide reactivity and nitric oxide binding, production and purification means and solubility. Such modifications will be known by those skilled in the art and may be incorporated into the modified human haemoglobin proteins as defined in claim 1.
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin beta chain subunit, wherein the at least one further modification is βL96Y.
In certain embodiments, the modified human haemoglobin protein further comprises at least one human haemoglobin alpha chain subunit, wherein the at least one further modification is αL91Y. In certain embodiments, the at least one further modification is αL29F.
In certain embodiments, the modified human haemoglobin protein further comprises at least one human haemoglobin alpha chain subunit, wherein the at least one further modification is a plurality of modifications selected from one or more of αL91Y and/or αL29F or a combination thereof.
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin gamma 1 chain subunit, wherein the at least one further modification is γ1V67F.
In certain embodiment, the modified human haemoglobin protein comprises at least one human haemoglobin gamma 2 chain subunit, wherein the at least one further modification is γ2V67F.
In certain embodiments, the modified human haemoglobin protein as defined in claim 1 may include at least one further modification such as but not limited to those disclosed in WO2009/004309 . Without being bound by theory such further modifications may introduce or enhance reduction of at least one tetravalent cation to a trivalent cation as compared to a reference protein. For example, the reduction of a ferryl (Fe⁴⁺) ion to a ferric (Fe³⁺) ion as is disclosed in WO2009/004309 . In these embodiments, the modified human haemoglobin protein may have decreased toxicity as compared to the reference protein.
In certain embodiments, the modified human haemoglobin protein as defined in claim 1 may include at least one further modification such as but not limited to a substitution of the most N-terminal amino acid residue with a methionine residue.
In certain embodiments, the modified human haemoglobin protein further comprises at least one further modification which reduces nitric oxide reactivity of the modified protein.
In certain embodiments, the modified human haemoglobin protein further comprises a plurality of further modifications.
In certain embodiments, the modified human haemoglobin protein is a human haemoglobin beta chain subunit and may comprise one or more further modifications listed below:
NA1(Val>Met); B13(Leu>Phe or Trp); G12(Leu>Phe or Trp); B10(Leu>Phe) and E4(Val>Leu); B10(Leu>Trp) and E4(Val>Leu); B14(Leu>Phe or Trp); G8(Leu>Phe) and G12(Leu>Trp); E11 (Val>Leu) and G8(Leu>Trp); E11 (Val>Trp) and G8(Leu>Met); E11 (Val>Leu) and G8(Leu>Phe); E11 (Val>Leu) and G8(Leu>Met); E11(Val>Phe) and G8(Leu>lle); E11(Val>Phe) and G8(Leu>Phe); E11(Val>Phe) and G8(Leu>Trp); E11(Val>Phe) and G8(Leu>Met); E11 (Val>Met) and G8(Leu>Trp); E11 (Val>Met) and G8(Leu>Trp) and E7(His>Gln); E11 (Val>Trp) and G8(Leu>lle); E7(His>Gln) and E11(Val>Trp); E7(His>Gln) and E11 (Val>Leu); E7(His>Gln) and E11 (Val>Phe); E7(His>Gln) and E11 (Val>Phe) and G8(Leu>Phe or Trp); E7(His>Gln) and E11(Val>Leu or Trp) and G8(Leu>Phe or Trp); E11 (Val>Trp or Phe) and G12(Leu>Trp or Met); E11(Val>Trp or Phe) and B13(Leu>Trp or Met); B10(Leu>Trp) and B13(Leu>Trp or Met); B10(Leu>Phe) and B13(Leu>Trp); B10(Leu>Trp or Phe) and G12(Leu>Trp); B10(Leu>Phe) and G12(Leu>Met); G8(Leu>Trp) and G12(Leu>Trp or Met); or G8(Leu>Trp) and B13(Leu>Trp or Met); C7 (Phe>Tyr).
The numbering used above is based on helix chain numbering, which can be cross-referenced to the primary sequence numbering of Table 2 below for the human haemoglobin A_o beta chain subunits. It will be understood by those skilled in the art that haemoglobin subunit proteins may be numbered by reference to individual helices or inter-helix residues as is set out in Table 2. For example, the F1 residue of the human haemoglobin beta chain subunit may be equivalent to the F1 residue in other haemoglobin beta chain subunits. It will be understood by those skilled in the art that at least one or more of the further modifications at equivalent positions in other modified human haemoglobin proteins of the present invention may also be included.
Without being bound by theory, the at least one modification enhances an electron transfer pathway to the at least one iron ion associated with the modified human haemoglobin protein via the at least one tyrosine residue of the modified human haemoglobin protein. This electron transfer pathway via the tyrosine residue of the modified protein may have a higher affinity than a direct electron transfer pathway to the at least one iron ion and so can result in more rapid reduction of the at least one iron ion.
In certain embodiments, the modified human haemoglobin protein is conjugated to at least one non-antigenic moiety. In certain embodiments, the non-antigenic moiety is conjugated to the modified human haemoglobin protein in order to help improve solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability. Such conjugates may also help to reduce clearance (e.g. renal clearance) of proteins. As used herein the term "conjugated" refers to a physical attachment of one identifiable moiety to another. A number of suitable non-antigenic moieties will be known by those skilled in the art.
In certain embodiments, the moiety may be a protein. In certain embodiments, wherein the moiety is a protein, the protein moiety may be produced as a fusion protein with the modified protein. Alternatively, the protein moiety and the modified protein may be expressed separately or co-expressed and linked by chemical means such as by a chemical cross linker. Suitable chemical cross linkers will be known by those skilled in the art. By way of example cross linking agents may be one or more of glutaraldehyde, disparin derivatives, polyaldehydes, diphosphate esters, triphosphate esters.
In certain embodiments, the protein moiety is an antioxidant enzyme. By way of example the antioxidant enzyme may be a catalase and/or superoxide dismutase. In certain embodiments, the protein moiety is a human catalase and/or human superoxide dismutase.
In certain embodiments, the at least one non-antigenic moiety is at least one polymeric moiety. In certain embodiments, the polymeric moiety is water-soluble, non-toxic and pharmaceutically inert. In certain embodiments, the polymeric moiety is at least one polyalkylene glycol. In certain embodiments, the polymeric moiety is polyethylene glycol (PEG).
In certain embodiments, the polymeric moiety can be covalently bound through amino acid residues via a reactive group, such as, a free amino, carboxyl group or sulfhydryl group. Reactive groups are those to which an activated PEG molecule can be bound. Examples of naturally occurring amino acid residues having a free amino group include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups (e.g., on cysteine) can also be used as a reactive group for attaching for example polyethylene glycol molecules.
The polymeric moiety used can be of any molecular weight,and can be branched or unbranched. In certain embodiments, the polyalkylene glycol has a molecular weight between 1000 Daltons and 100,000 Da. For example, the polyalkylene glycol can have an average molecular weight of 1000, 5000, 10000, 15000, 20000, 25000, 30000, 35000, 40000, 50000, 60000, 70000, 80000, 90000 or 100000 Da.
The number of polymeric moieties attached to each modified protein (i.e. number of PEG molecules) can also vary. For example, the modified protein may be linked, on average, to 1, 2, 3, 4, or 5, or more polyethylene glycol molecules.
In certain embodiments of the present invention there is provided a multimeric protein comprising at least one modified human haemoglobin protein as defined in claim 1. In certain embodiments, multimeric forms of the modified human haemoglobin protein may prolong circulation lifetime of the modified human haemoglobin protein, improve a rate at which an oxidised form of the modified human haemoglobin protein is capable of re-oxygenation to an oxygen-binding form as compared to the reference protein, improve oxygen-carrying properties and/or reduce side-effects.
In certain embodiments, the modified human haemoglobin protein is a dimer. In certain embodiments, the modified human haemoglobin protein is a trimer. In certain embodiments, the modified human haemoglobin protein is a tetramer.
In certain embodiments, the multimeric protein comprises at least one or more reference oxygen-carrying proteins and/or reference oxygen-carrying protein subunits. In certain embodiments, the at least one reference oxygen-carrying protein and/or reference oxygen-carrying protein subunit may include at least one further modification as described herein.
These further modifications may affect properties of each of the at least one reference oxygen-carrying proteins and/or reference oxygen-carrying protein subunits such as oxygen affinity or cooperativity, stability and assembly rate, decreased porphyrin loss, decreased metallic ion autoxidation rate, resistance to proteolytic degradation, decreased aggregation, nitric oxide reactivity and nitric oxide binding, production and purification means and solubility. Such modifications will be known by those skilled in the art.
By way of example further modifications that may be included wherein the in the at least one reference oxygen-carrying protein and/or oxygen-carrying protein subunit comprises a haemoglobin alpha chain subunit may include:
NA1(Val>Met); E11(Val>Leu) and E7(His>Gln); E11(Val>Phe or Trp) and E7(His>Gln); E11(Val>Phe or Trp or Leu) and E7(His>Gln) and G8(Leu>Phe or Trp); B10(Leu>Phe) and E4(Val>Leu); 0 B10(Leu>Trp) and E4(Val>Leu); B10(Leu>Trp) and E7(His>Gln); B10(Leu>Trp) and E11(Val>Phe); B10(Leu>Trp) and E11 (Val>Trp); B10(Leu>Trp) and E11(Val>Leu) and G8(Leu>Trp); B10(Leu>Trp) and E11 (Val>Leu) and G8(Leu>Phe); B10(Leu>Trp) and E11(Val>Phe) and G8(Leu>Trp); B10(Leu>Trp) and E11(Val>Phe) and G8(Leu>llc); B10(Leu>Trp) and E7(His>Gln) and E11(Val>Leu) and G8(Leu>Trp); B10(Leu>Trp) and E11 (Val>Trp) and G8(Leu>Trp); E11 (Val>Leu) and G8(Leu>Phe); E11 (Val>Leu) and G8(Leu>Trp); B13(Met>Phe or Trp); G12(Leu>Phe or Trp); or B14(Phe>Trp).
The numbering used above is based on helix chain numbering, which can be cross-referenced to the primary sequence numbering of Table 2 below for the human haemoglobin alpha chain subunits. It will be understood by those skilled in the art that at least one or more of these further modifications at equivalent positions in other oxygen-carrying proteins of the invention may also be included.
Thus in certain embodiments wherein the modified human haemoglobin protein of the present invention is a human haemoglobin beta chain subunit the multimeric protein may comprise a tetrameric haemoglobin protein comprising two modified human haemoglobin beta chain subunits as defined in claim 1 and two wild-type haemoglobin alpha chain subunits or two reference alpha chain subunits e.g. αV1M haemoglobin alpha chain subunits. In certain embodiments, the multimeric protein may comprise any number or combination of modified human haemoglobin proteins as defined in claim 1 and any number or combination of reference proteins (e.g. haemoglobin chain subunits including the further modifications V1M and G1M and/or wild-type haemoglobin chain subunits) as described herein. For example, the multimer may be any one of the tetrameric haemoglobins given in Table 1 wherein at least one chain subunit is a modified human haemoglobin protein as defined in claim 1.
In certain embodiments, the modified human haemoglobin protein is adult haemoglobin (also referred to as Haemoglobin A, HbA, or α₂β₂) comprising 2 alpha chain subunits and two beta chain subunits.
In certain embodiments, wherein the modified human haemoglobin protein is a multimer, one or more modifications and/or further modifications as described herein may be located in a single subunit or may be distributed through two, three or four different subunits
In certain embodiments, the modified human haemoglobin protein is a foetal haemoglobin (also referred to as haemoglobin F, HbF, and/or α₂γ₂). Foetal haemoglobin comprises 2 alpha chain subunits and 2 gamma chain subunits. Aptly the gamma chain subunits may be gamma 1 or gamma 2.
In certain embodiments, the modified human haemoglobin protein comprises a foetal haemoglobin comprising at least one haemoglobin gamma1 and/or gamma 2 chain subunit comprising the modification γL96Y.
In certain embodiments, the modified human haemoglobin protein comprises a foetal haemoglobin comprising the at least one modification γL96Y and a further modification selected from αV1M and/or γG1M or a combination thereof.
In certain embodiments, the modified human haemoglobin protein comprises a foetal haemoglobin comprising the modifications, αL91Y and γL96Y and a further modification selected from αV1M and γG1M or a combination thereof.
In certain embodiments, the modified human haemoglobin protein comprises a foetal haemoglobin comprising the modifications αL29F, γV67F and γL96Y or a combination thereof and a further modification selected from αV1M and γG1M or a combination thereof.
In certain embodiments, the modified human haemoglobin protein comprises a foetal haemoglobin comprising the modifications αL29F, αL91Y, γV67F and γL96Y or a combination thereof and the further modifications αV1M and γG1M or a combination thereof.
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin alpha chain subunit as set forth in SEQ. ID. NO. 6 (αV1M reference) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified yG1M). Aptly the modified human haemoglobin protein comprises two human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 12 (αL91Y modified) and two human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified yG1M).
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 11 (αL91Y modified, V1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified yG1M). Aptly the modified human haemoglobin protein comprises two human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 11 (αL91Y modified, V1M) and two human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified γG1M).
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 14 (αL29F modified αV1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 15 (γV67F modified γG1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified γG1M). Aptly the modified human haemoglobin protein comprises two human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 14 (αL29F modified αV1M) and one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 15 (γV67F modified γG1M) and one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified γG1M).
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 14 (αL29F modified αV1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 17 (γV67F, γL96Y modified γG1M). Aptly the modified human haemoglobin protein comprises two human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 14 (αL29F modified αV1M) and two human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 17 (γV67F, γL96Y modified yG1M).
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 14 (αL29F modified αV1M) and at least one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 11 (αL91Y modified αV1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 15 (γV67F modified yG1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified γG1M). Aptly the modified human haemoglobin protein comprises one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 14 (αL29F modified αV1M) and one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 11 (αL91Y modified αV1M) and one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 15 (γV67F modified γG1M) and one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 13 (γL96Y modified yG1M).
In certain embodiments, the modified human haemoglobin protein comprises at least one human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 16 (αL29F, αL91Y modified αV1M) and at least one human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 17 (γV67F, γL96Y modified yG1M). Aptly the modified human haemoglobin protein comprises two human haemoglobin alpha chain subunits as set forth in SEQ. ID. NO. 16 (αL29F, αL91Y modified αV1M ) and two human haemoglobin gamma 2 chain subunit as set forth in SEQ. ID. NO. 17 (γV67F, γL96Y modified γG1M).
In certain embodiments the multimer and/or multimeric protein is cross linked. Methods of cross linking proteins will be known by those skilled in the art but by way of example suitable cross linking method may include but are not limited to chemical cross lining and fusion protein recombinant expression.
Without being bound by theory, the enhanced reduction activity increases the reduction of a ferric (Fe³⁺) ion of a non-functional (non-oxygen binding) met-haemoglobin form to a functional (oxygen binding) oxy-haemoglobin form wherein the iron ion is a ferrous (Fe²⁺) ion. Thus, the at least one modification as defined in claim 1 may help to increase the rate at which an oxygen carrying and/or binding form of a haemoglobin and/or haemoglobin chain subunit is formed.
In certain embodiments, the at least one modification is located on the EF helix.
In certain embodiments, the at least one modification is located on the F helix.
In certain embodiments, the at least one modification is residue EF8 (Thr>Tyr).
In certain embodiments, the at least one modification is helical residue F1 (Phe>Tyr).

The numbering used above to define the location of certain modifications refers to the helix chain positions for human haemoglobin alpha and beta chain subunits as is given in Table 2. It will be understood by those skilled in the art that the helical residue positions may apply to other human haemoglobin chain subunits of embodiments of the present invention.

Table 2: Amino Acid Sequence and Helical Residue Notation for Human Haemoglobin A_o

Helix	α	Helix	β	Helix	α	Helix	β
NA1	1 Val	NA1	1 Val	E17	68 Asn	E17	73 Asp
-	-	NA2	2 His	E18	69 Ala	E18	74 Gly
NA2	2 Leu	NA3	3 Leu	E19	70 Val	E19	75 Leu
A1	3 Ser	A1	4 Thr	E20	71 Ala	E20	76 Ala
A2	4 Pro	A2	5 Pro	EF1	72 His	EF1	77 His
A3	5 Ala	A3	6 Glu	EF2	73 Val	EF2	78 Leu
A4	6 Asp	A4	7 Glu	EF3	74 Asp	EF3	79 Asp
A5	7 Lys	A5	8 Lys	EF4	75 Asp	EF4	80 Asn
A6	8 Thr	A6	9 Ser	EF5	76 Met	EF5	81 Leu
A7	9 Asn	A7	10 Ala	EF6	77 Pro	EF6	82 Lys
A8	10 Val	A8	11 Val	EF7	78 Asn	EF7	83 Gly
A9	11 Lys	A9	12 Thr	EF8	79 Ala	EF8	84 Thr
A10	12 Ala	A10	13 Ala	F1	80 Leu	F1	85 Phe
A11	13 Ala	A11	14 Leu	F2	81 Ser	F2	86 Ala
A12	14 Trp	A12	15 Trp	F3	82 Ala	F3	87 Thr
A13	15 Gly	A13	16 Gly	F4	83 Leu	F4	88 Leu
A14	16 Lys	A14	17 Lys	F5	84 Ser	F5	89 Ser
A15	17 Val	A15	18 Val	F6	85 Asp	F6	90 Glu
A16	18 Gly	-	-	F7	86 Leu	F7	91 Leu
AB1	19 Ala	-	-	F8	87 His	F8	92 His
B1	20 His	B1	19 Asn	F9	88 Ala	F9	93 Cys
B2	21 Ala	B2	20 Val	FG1	89 His	FG1	94 Asp
B3	22 Gly	B3	21 Asp	FG2	90 Lys	FG2	95 Lys
B4	23 Glu	B4	22 Glu	FG3	91 Leu	FG3	96 Leu
B5	24 Tyr	B5	23 Val	FG4	92 Arg	GF4	97 His
B6	25 Gly	B6	24 Gly	FG5	93 Val	FG5	98 Val
B7	26 Ala	B7	25 Gly	G1	94 Asp	G1	99 Asp
B8	27 Glu	B8	26 Glu	G2	95 Pro	G2	100 Pro
B9	28 Ala	B9	27 Ala	G3	96 Val	G3	101 Glu
B10	29 Leu	B10	28 Leu	G4	97 Asn	G4	102 Asn
B11	30 Glu	B11	29 Gly	G5	98 Phe	G5	103 Phe
B12	31 Arg	B12	30 Arg	G6	99 Lys	G6	104 Arg
B13	32 Met	B13	31 Leu	G7	100 Leu	G7	105 Leu
B14	33 Phe	B14	32 Leu	G8	101 Leu	G8	106 Leu
B15	34 Leu	B15	33 Val	G9	102 Ser	G9	107 Gly
B16	35 Ser	B16	34 Val	G10	103 His	G10	108 Asn
C1	36 Phe	C1	35 Tyr	G11	104 Cys	G11	109 Val
C2	37 Pro	C2	36 Pro	G12	105 Leu	G12	110 Leu
C3	38 Thr	C3	37 Trp	G13	106 Leu	G13	111 Val
C4	39 Thr	C4	38 Thr	G14	107 Val	G14	112 Cys
C5	40 Lys	C5	39 Gln	G15	108 Thr	G15	113 Val
C6	41 Thr	C6	40 Arg	G16	109 Leu	G16	114 Leu
C7	42 Tyr	C7	41 Phe	G17	110 Ala	G17	115 Ala
CE1	43 Phe	CD1	42 Phe	G18	111 Ala	G18	116 His
CE2	44 Pro	CD2	43 Glu	G19	112 His	G19	117 His
CE3	45 His	CD3	44 Ser	GH1	113 Leu	GH1	118 Phe
CE4	46 Phe	CD4	45 Phe	GH2	114 Pro	GH2	119 Gly
-	-	CD5	46 Gly	GH3	115 Ala	GH3	120 Lys
CE5	47 Asp	CD6	47 Asp	GH4	116 Glu	GH4	121 Glu
CE6	48 Leu	CD7	48 Leu	GH5	117 Phe	GH5	122 Phe
CE7	49 Ser	CD8	49 Ser	H1	118 Thr	H1	123 Thr
CE8	50 His	D1	50 Thr	H2	119 Pro	H2	124 Pro
-	-	D2	51 Pro	H3	120 Ala	H3	125 Pro
-	-	D3	52 Asp	H4	121 Val	H4	126 Val
-	-	D4	53 Ala	H5	122 His	H5	127 Gln
-	-	D5	54 Val	H6	123 Ala	H6	128 Ala
-	-	D6	55 Met	H7	124 Ser	H7	129 Ala
CE9	51 Gly	D7	56 Gly	H8	125 Leu	H8	130 Tyr
E1	52 Ser	E1	57 Asn	H9	126 Asp	H9	131 Gln
E2	53 Ala	E2	58 Pro	H10	127 Lys	H10	132 Lys
E3	54 Gln	E3	59 Lys	H11	128 Phe	H11	133 Val
E4	55 Val	E4	60 Val	H12	129 Leu	H12	134 Val
E5	56 Lys	E5	61 Lys	H13	130 Ala	H13	135 Ala
E6	57 Gly	E6	62 Ala	H14	131 Ser	H14	136 Gly
E7	58 His	E7	63 His	H15	132 Val	H15	137 Val
E8	50 Gly	E8	64 Gly	H16	133 Ser	H16	138 Ala
E9	60 Lys	E9	65 Lys	H17	134 Thr	H17	139 Asn
E10	61 Lys	E10	66 Lys	H18	135 Val	H18	140 Ala
E11	62 Val	E11	67 Val	H19	136 Leu	H19	141 Leu
E12	63 Ala	E12	68 Leu	H20	137 Thr	H20	142 Ala
E13	64 Asp	E13	69 Gly	H21	138 Ser	H21	143 His
E14	65 Ala	E14	70 Ala	HC1	139 Lys	HC1	144 Lys
E15	66 Leu	E15	71 Phe	HC2	140 Tyr	HC2	145 Tyr
E16	67 Thr	E16	72 Ser	HC3	141 Arg	HC3	146 His

In certain embodiments, wherein the modified human haemoglobin protein is a haemoglobin beta chain subunit, the at least one modification is βT84Y.
In certain embodiments, the at least one modification is βF85Y.
In certain embodiments, the at least one modification is a plurality of modifications selected from βF85Y and/or βT84Y or a combination thereof.
In certain embodiments, wherein the modified human haemoglobin protein is a haemoglobin gamma 1 chain subunit, the at least one modification is γ1L96Y.
In certain embodiments, the at least one modification is a plurality of modifications selected from γ1L96Y and γ1V67F.
In certain embodiments, wherein the modified human haemoglobin protein is a haemoglobin gamma 2 chain subunit, the at least one modification is γ2L96Y.
In certain embodiments, the at least one modification is a plurality of modifications selected from γ2L96Y and γ2V67F.
The numbering used above for certain embodiments of the present invention wherein the modified human haemoglobin protein is a haemoglobin chain subunit refers to the amino acid residue positions with reference to the wild-type human haemoglobin beta, alpha, gamma 1 and gamma 2 chain subunit amino acid sequences as set forth in SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3 and SEQ. ID. NO. 4 respectively. It will be understood by those skilled in the art that in certain embodiments wherein the modified protein comprises further modifications such as deletions or insertions the numbering of the above-mentioned modifications will change. By way of example if the N-terminus amino acid residue is deleted the modification βT84Y would change to be βT83Y and so on. If an amino acid residue is inserted at the N-terminus of the modified protein the modification βT84Y would be denoted as βT85Y. For example in certain embodiments the N-terminal of the protein may include a methionine residue encoded by the start codon which is usually cleaved from the mature protein, in such embodiments a T84Y modification would be denoted as T85Y, a F85Y modification would be denoted F86Y and a L91Y modification would be denoted L92Y and so on.
In one aspect of the present invention there is provided a composition comprising,
a modified human haemoglobin protein as defined in any one of claims 1 to 5; and
a pharmaceutically acceptable carrier or diluents.
In certain embodiments, the composition further comprises at least one reductant. In certain embodiments, the at least one reductant is for donating at least one electron so as to reduce the at least one metallic iron ion.
In certain embodiments, the at least one reductant is ascorbate. In certain embodiments, the at least one reductant is Nicotinamide adenine dinucleotide phosphate (NADPH). In certain embodiments, the at least one reductant is Nicotinamide Adenine Dinucleotide (NADH). In certain embodiments, the reductant is one or more of ascorbate, NADP and/or NADH. Other suitable reductants will be known to those skilled in the art.
In certain embodiments, the composition is a pharmaceutical composition and is for administration to a subject. In certain embodiments, the subject is a mammalian subject. In certain embodiments, the subject is a human.
In certain embodiments, the composition is a blood substitute composition. A blood substitute composition is a composition which may be used to mimic and/or fulfil the functions of blood. Blood substitute compositions may include such components as plasma, serum albumin and other fluids of which are not derived from blood such as plasma volume expanders; these, may include for example crystalloid intravenous solutions. Other suitable blood substitute components will be known to those skilled in the art. The components of a blood substitute that is able to mimic bloods ability to carry and transfer oxygen may be referred to as an oxygen therapeutic. Thus, in certain embodiments the modified human haemoglobin protein and compositions thereof, of the present invention may be referred to as oxygen therapeutics.
In certain embodiments, the composition is a resuscitation fluid. Resuscitation fluids are fluids that may be used to restore intravascular volume. Without being bound by theory resuscitation fluids may be broadly categorized into two main categories, colloid and crystalloid solutions. Colloid solutions are suspensions of molecules within a carrier solution that are relatively incapable of crossing a healthy semipermeable capillary membrane owing to the molecular weight of the molecules. Crystalloids are solutions of ions that are freely permeable but contain concentrations of salts such as sodium and/or chloride that determine the tonicity of the fluid. By way of example resuscitation fluids may include at least one or more of sodium, potassium, calcium, magnesium, chloride, acetate, lactate, malate, gluconate, bicarbonate or octanoate. Other suitable resuscitation fluid components will be known by those skilled in the art.
In one aspect of the present invention there is provided a modified human haemoglobin protein as defined in any one of claims 1 to 5 or composition as defined in any one of claims 6 to 8 for use as a medicament.
Further details of the modified human haemoglobin protein are provided herein.
The modified human haemoglobin proteins and compositions thereof may be for use as an oxygen therapeutic. As used herein the term "oxygen therapeutic" refers to a molecule that is able to transport and release oxygen. Oxygen therapeutics may be used as part of a blood substitute or may be referred to as a blood substitute themselves by those of ordinary skill in the art.
Modified human haemoglobin proteins and compositions thereof of embodiments the present invention may be for use in conditions wherein there is a need for the restoration, maintenance or replacement of oxygen. These include but are not limited to ischemia (such as ischemia induced by heart attack, stroke or cerebrovascular trauma).
In certain embodiments, the modified human haemoglobin proteins and compositions thereof as described herein are for use in the treatment of ischemia.
In certain embodiments, the modified human haemoglobin proteins and compositions thereof as described herein are for use in the treatment and/or prevention of hypoxia.
In certain embodiments, the modified human haemoglobin proteins and compositions thereof as described herein are for use in the treatment and/or prevention of ischemia and/or hypoxia.
Ischemia is a lack of and/or reduced blood flow to an organ or tissue. Ischemia may be caused by a blockage within one or more blood vessels or due to external compression of one or more blood vessels. By way of example a blockage within a blood vessel may be a thrombus or atherosclerosis. Such blockages may be arterial blockages or venous blockages, other blockages will be known by those skilled in the art and may cause what is known in the art as arterial or venous insufficiency. By way of example external compression of a blood vessel may be caused by trauma which may induce swelling and/or inflammation therefore constricting the blood vessels or may be caused by an external object and/or internal tissue such as a tumour or cancerous growth or inflamed organ applying pressure to a blood vessel. Ischemia may also occur when blood loss occurs such as due to acute haemorrhage, due to trauma or during surgical procedures. Types of ischemia will be known by those skilled in the art but non-limiting examples include myocardial ischemia, cerebral ischemia, limb ischemia, mesenteric ischemia and/or cutaneous ischemia.
As red blood cells normally carry 98% of the oxygen in blood to cells, tissues and organs, ischemia may lead to hypoxia. Hypoxia is a lack of and/or reduced amount of oxygen being transported to cells, tissues or organs and may be defined as a decrease in the oxygen tension within a tissue below normal functioning levels. Oxygen tension is a measure of the partial pressure of oxygen within blood and/or a tissue. Oxygen transfer from blood vessels, such as a capillary to associated tissue or cells may be characterised in terms of oxygen flux. As used herein the term "oxygen flux" refers to the mass of oxygen transported through a blood vessel per unit of time. When blood flow is reduced as may be caused for example by blood loss (haemorrhage), ischemia and/or shock (e.g. volume deficiency shock, anaphylactic shock, septic shock or allergic shock) a reduced amount of red blood cells flow through the blood vessel and therefore the oxygen flux decreases, therefore leading to a decrease in the transfer of oxygen to associated cells or tissue thereby resulting in hypoxia and in some cases anoxia, which is characterised as a tissue condition wherein no measurable oxygen is present. Both hypoxia and anoxia may lead to death of cells and/or tissue (necrosis). Thus certain embodiments of the modified proteins and compositions thereof, of the present invention may be for use in the treatment and/or prevention of hypoxia and/or anoxia and therefore may be for use in the prevention of necrosis.
Certain embodiments of the modified human haemoglobin proteins and compositions described herein may be for use as a bridge to red blood cell transfusion. The term "bridge to red blood cell transfusion" as used herein refers to when red blood cell transfusion is a viable treatment but is delayed. Therefore the use of certain embodiments of the modified human haemoglobin protein and compositions thereof described herein may help to prevent and/or treat ischemia and/or hypoxia that may occur, until a red blood cell transfusion can be performed. By way of example certain embodiments of the modified human haemoglobin proteins and compositions thereof may be used in situations when no red blood cells are readily available, such as on a battlefield or in remote areas and/or when suitable red blood cells cannot be readily matched to the blood type of a subject in need thereof or when amounts of red blood cells are not sufficient for treatment such as when treating large numbers of subjects in need thereof.
The terms "patient", "subject" and "individual" may be used interchangeably and refer to either a humans or non-human mammal. In certain embodiments, the subject is a human.
The therapeutically effective amount of the modified proteins and compositions as described herein will depend on the route of administration, the type of subject being treated, and the physical characteristics of the specific subject under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. Certain embodiments of the modified proteins and compositions thereof of the present disclosure may be particularly useful for use in the treatment of humans.
An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the person skilled in the art.
The term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used. pH buffering agents may be phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine, or acetate or mixtures thereof. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans.
"Treatment" is an approach for obtaining beneficial or desired clinical results. For the purposes of the present disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treatment" is an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures in certain embodiments. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. By treatment is meant inhibiting or reducing an increase in pathology or symptoms when compared to the absence of treatment and is not necessarily meant to imply complete cessation of the relevant condition.
Certain embodiments of the modified proteins and compositions described herein may be for use in cell, tissue, or organ culturing and/or preservation. In certain embodiments, the modified proteins and compositions thereof may be used alone or in addition to one or more further oxygen carrying proteins and/or in addition to a culture and/or preservation media suitable for cell culture, tissue culture and/or organ culture and/or tissue and/or organ perfusion. Without being bound by theory, certain embodiments of the modified proteins as described herein may help to increase the oxygen transported to said cells, tissues and/or organs and therefore increase the probability of maintaining healthy normal living cells, tissue or organs.
In certain embodiments, the modified proteins and compositions described herein may also extend the lifetime of cultured cells, tissues or organs. Thus, in certain embodiments, there is provided a composition which comprises a modified protein described herein and a cell culture media. In certain embodiments, the cell culture media is a liquid medium and may be selected from Viaspan ^® , 1 IGL ^® , Celsior ^® , SCOT Maco ^® , BMPS Belzer ^® , Custodiol ^® (HTK), Euro-Collins ^® , Soltran ^® , Perfadex ^® , Ringer lactate ^® and/or Plegisol ^® , Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12, minimal essential media, Roswell Park Memorial Institute medium 1640 or 199 and/or any medium composition suitable for preservation of organs, tissues, or organ cells or tissue, or suitable for organ or tissue perfusion.
Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to" and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and/or steps are mutually exclusive. The invention is not restricted to any details of any foregoing embodiments. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Examples

Materials and methods

Production of recombinant haemoglobin and modified proteins

The methods used were as performed as in "Reeder et al. Tyrosine Residues as Redox Cofactors in Human Haemoglobin: Implications For Engineering Nontoxic Blood Substitutes. J Biol Chem. 2008 ". A summary of the methods used is given below.
Genes from human haemoglobin (Hb) α and β chain subunits were optimised for expression in Escherichia Coli (E coli) and cloned into the vector pETDuet to form HbpETDuet.
Mutagenesis was performed by Epoch Life Sciences (Sugar land, Texas). Modified and unmodified plasmids were transformed into E coli BL21 DE3 cells using standard procedures. Resulting mutant clones were sequenced with BigDye terminator version 3.1 (Applied Biosystems Inc, Foster City, CA) to confirm correct sequences. E. coli BL21 DE3 cells harbouring the plasmid HbpETDuet encoding for a reference or modified Hb was grown in 2-liter Erlenmeyer flasks containing 1 litre of Terrific Broth medium with 100 µg/ml carbenicillin at 37 °C and 120 rpm until A ₆₂₀ ≥1. Expression of reference Hb or modified Hb was then induced by adding 0.1 mM isopropyl 1-thio-p-D-galactopyranoside, 0.3 mM δ-aminolevulinic acid, and CO gas. Culture conditions after induction were 22 °C and 60 rpm. Cells were harvested and suspended in 10 mM sodium phosphate buffer, pH 6.0, before sonication. Following centrifugation for 1 hour at 20,000 rpm, the supernatant was adjusted to pH 6.2 and filtrated using a 0.45-µm Minisart filter (Sartorius). The reference Hb or modified Hb was purified using ion exchange chromatography with CM-Sepharose FF column (GE Healthcare). After sample application, the column was washed with 10 mM sodium phosphate buffer, pH 6.0, until absorbance of eluted fractions returned to a base line absorbance value. The reference Hb or modified Hb was eluted with 70 mM sodium phosphate buffer, pH 7.2, and concentrated using Viva-Spin columns (Vivascience, 30-kDa molecular mass cutoff). The concentrated sample was then applied to a Sephacryl S-200 gel filtration column (GE Healthcare) using elution buffer on an ÄKTA purifier system. Globin-containing fractions were concentrated as above, flash-frozen in liquid nitrogen, and stored at -80 °C.
Prior to ferryl reduction experimentation, the reference Hb or modified Hb was oxidized to the ferric form by the addition of a 1.5 M excess potassium ferricyanide following CO removal by shining light on the sample with gentle oxygenation using a stream of oxygen gas. Ferriferrocyanide was removed by filtration through a Sephadex G-25 column (10 × 1 cm). Concentration of reference Hb or modified Hb was determined from reduction of an aliquot of the ferric Hb using sodium dithionite to the deoxy form.

Autoxidation measurements

The rate of conversion of ferrous oxy-haemoglobin to ferric met-haemoglobin was monitored by UV-visible spectroscopy. A 1 ml solution of 20 mM sodium phosphate (pH 7.4) with a protein concentration of 10 µM haem were studied at either 25ºC or 37ºC. For samples at 25ºC spectra between 375-700 nm were collected for up to 48 hours. For samples at 37ºC spectra between 375-700 nm were collected for up to 3 hours. Kinetic traces were analysed by fitting to single exponential fits.

Haem release from haemoglobin

Recombinant HbA (final concentration of 1.7 µM) was mixed with an excess of the haem binding protein hemopexin (final concentration 2 µM) at 37ºC in sodium phosphate buffer (20 mM, pH 7.2). The rate of change of haem from high spin in met-haemoglobin to low spin when bound by hemopexin was monitored by measuring optical changes in the Soret and visible regions of the optical spectra. The rate of release was monitored by measuring the increase in the difference between absorbance at 425 nm and 495 nm. Or the decrease in the difference between absorbance at 401 nm and 495 nm. Time courses were analysed by single exponential fits. Absorbance measurements were taken using an Agilent Cary 5000 spectrophotometer.

Ferrvl reduction measurements

Samples of ferryl haemoglobin were made by adding H₂O₂ and ferric met-haemoglobin in a 3:1 ratio and incubating for 10-15 minutes. Full conversion was confirmed by analysing optical spectra from the Soret and visible regions. A small concentration of catalase (1-5 nM) (Sigma-Aldrich) was added to remove any excess H₂O₂ (Sigma Aldrich). Seconds after addition of catalase, sodium ascorbate of varying concentrations was added to the sample and the reduction of ferryl haemoglobin to ferric met-haemoglobin was measured optically using the difference between absorbance measured at 535 nm and 630 nm (i.e. Abs₅₄₅ minus Abs₆₃₀). The time courses were fitted to double exponential fits assuming that full reduction was achieved half via α-chains and half via β-chains and forcing fits accordingly (i.e. an equal amplitude of absorbance for each of the chain types). The ascorbate concentration dependence of the pseudo-first order rate constants for α-chain and β-chain ferryl reduction were fitted to a double rectangular hyperbola, representing different electron transfer pathways. Buffer was 20 mM sodium phosphate pH 7.2, and experiments were performed at a temperature of 25 °C, concentration of haemoglobin used was 10 µM. Absorbance measurements were taken using an Agilent Cary 5000 spectrophotometer.

Ferric reduction measurements

Ferric Haemoglobin at a concentration of 20 µM in sodium phosphate buffer (20 mM, pH 7.2) was mixed with sodium ascorbate in a 1:1 volume to volume ratio (to give a final concentration of ferric haemoglobin of 10 µM) at 25ºC. The final concentration of sodium ascorbate was 0.1 mM, 1mM or 10 mM. The reaction mix was monitored optically using a Cary 5000 spectrophotometer (Agilent) for a period of 1 to 4 hours. The time courses of absorbance at 577-630 nm were fitted to a single exponential function minimising the least squares using Microsoft Excel Solver.
The percentage of oxy-haemoglobin formed was calculated by normalising change in absorbance against the expected change in absorbance for total conversion of met-haemoglobin to oxy-haemoglobin.

Results

Autoxidation

The rate of autoxidation of ferrous oxy-haemoglobin can be seen for wild-type (wt) recombinant HbA protein, a βT84Y modified recombinant HbA protein, reference (V1M modified wild-type protein) recombinant HbA protein and a βT84Y (V1M) modified recombinant HbA protein in Figure 1. Introducing a βT84Y modification can be seen to not result in increased autoxidation as compared to wild-type and V1M modified reference proteins. This indicates that the βT84Y modification results in a relatively stable modified protein that does not readily autoxidise. This further indicates therefore that a the βT84Y modified proteins may be less likely to autoxidise when in use.

Haem release from haemoglobin

The rate of haem release from the met forms of wild-type (wt) recombinant HbA protein, a βT84Y modified recombinant HbA protein, a reference (V1M modified wild-type (wt(V1M)) recombinant HbA protein and a βT84Y (V1M) modified recombinant HbA protein can be observed in Figure 2. No significant difference in the rate of haem loss can be observed for both βT84Y modified proteins as compared to wildtype and reference V1M modified proteins. This indicates that the βT84Y modification does not reduce binding of the haem group and so indicates that βT84Y modified proteins will retain binding of its haem cofactor when in use.

Ferryl reduction

The percentage of ferryl haemoglobin reduced to ferric met-haemoglobin after 1 minute incubation with 30 µM ascorbate increases above that of the wild-type protein for all modified proteins studied (Figure 3). This observation indicates that all the modified proteins studied are able to be reduced to the less toxic form of ferric met-haemoglobin under physiological conditions.

Ferric reduction

The percentage of ferrous oxy-haemoglobin formed in the presence of 100 µM ascorbate over a time period of 60 minutes is observed to be increased for the βT84Y, βF85Y and αL91Y modified rHbA proteins in comparison to a wild-type protein as well as a number of other modified proteins that display ferryl haemoglobin to ferric met-haemoglobin reduction (Figure 4).
The ability of the βT84Y modified protein to reduce ferric met-haemoglobin to ferrous oxy-haemoglobin is further shown by Figure 5. It can be seen that in comparison to a wild-type protein that the rate of ferric oxy-haemoglobin production is greater than that of the wild-type protein (right hand graph) as is shown by the increased absorbance observed in the region for ferric oxy-haemoglobin for the βT84Y modified protein.
When the percentage of reduction of ferryl haemoglobin reduction to ferric met-haemoglobin reduction is plotted against the percentage of ferric met-haemoglobin to ferrous oxy-haemoglobin there is no correlation seen as is shown by Figure 6.
Mutations in foetal haemoglobin are also able to enhance the reduction of ferric met-haemoglobin to ferrous oxy-haemoglobin. For example, the mutations αL91Y, γF85Y and γL96y show a significantly increased rate constant for ferric haem reduction by the external reductant ascorbate (Figure 12).
These modifications also function to enhance ferric haem reduction in the presence of additional mutations designed to improve protein production (G1M and/or V1M) or decrease NO scavenging (αL29F, γV67F). It can be seen from Figure 7 that for modified proteins containing one or more haemoglobin chain subunits comprising one or more of the modifications αL29F, αL91Y, γV67F and γL96y with and without a further modification (G1M and/or V1M) all show improved percentage of oxy-haemoglobin formed from met-haemoglobin in comparison to both wild-type and/or reference proteins. This indicates that the modifications singularly or in combination enhance or introduce reduction of haem group metal ions leading to formation of oxy-haemoglobin.

Claims

A modified human haemoglobin protein comprising at least one modified human haemoglobin chain subunit, wherein the subunit comprises at least one substitution of an amino acid residue with at least one tyrosine residue,
wherein said substitution enhances reduction of at least one iron ion and increases a rate at which an oxidised form of the modified human haemoglobin protein is capable of re-oxygenation to an oxygen-binding form as compared to a reference protein,

wherein the at least one iron ion is located within at least one haem bound to the modified human haemoglobin protein and is associated with the modified human haemoglobin protein and the modified human haemoglobin protein exhibits enhanced reduction of a ferric (Fe³⁺) ion to a ferrous (Fe²⁺) ion as compared to a reference protein,

wherein the modified human haemoglobin chain subunit is selected from at least one or more of a beta or a gamma chain subunit and the substitution is selected from one or more of:
a. βT84Y, βF85Y mutation or a combination thereof, wherein the numbering refers to the amino acid residue positions with reference to the wild-type human haemoglobin beta chain subunit amino acid sequence as set forth in SEQ ID NO: 1;

b. γ1F85Y, wherein the numbering refers to the amino acid residue positions with reference to the wild-type human haemoglobin gamma 1 chain subunit amino acid sequences as set forth in SEQ ID NO: 3;

c. γ2F85Y, wherein the numbering refers to the amino acid residue positions with reference to the wild-type human haemoglobin gamma 2 chain subunit amino acid sequence as set forth in SEQ ID NO: 4;

d. γ1L96Y, wherein the numbering refers to the amino acid residue positions with reference to the wild-type human haemoglobin gamma 1 chain subunit amino acid sequences as set forth in SEQ ID NO: 3; and/or

e. γ2L96Y, wherein the numbering refers to the amino acid residue positions with reference to the wild-type human haemoglobin gamma 2 chain subunit amino acid sequence as set forth in SEQ ID NO: 4;

and wherein the reference protein is:
a wild-type version of the modified human haemoglobin protein,

a wild-type version of the human haemoglobin beta chain subunit with an amino acid sequence as set forth in SEQ ID NO:1 or a human haemoglobin beta chain subunit in which the first amino acid residue (valine) of the wild-type human haemoglobin beta chain subunit has been substituted with a methionine as set forth in SEQ. ID. No. 5 (βV1M),

a wild-type version of the human haemoglobin gamma 1 chain subunit with an amino acid sequence set forth in SEQ ID NO: 3 or a human haemoglobin gamma 1 chain subunit in which the first amino acid residue (glycine) of the wild-type human haemoglobin gamma 1 chain subunit has been substituted with a methionine as set forth in SEQ. ID. No. 7 (γ1G1M), or

a wild-type version of the human haemoglobin gamma 2 chain subunit with an amino acid sequence as set forth in SEQ ID NO: 4 or a human haemoglobin gamma 2 chain subunit in which the first amino acid residue (glycine) of the wild-type human haemoglobin gamma 2 chain subunit has been substituted with a methionine as set forth in SEQ. ID. No. 8 (γ2G1M).
The modified human haemoglobin protein of claim 1, wherein the modified human haemoglobin protein is conjugated to at least one protecting group.
The modified human haemoglobin protein according to claim 2, wherein the at least one protecting group is at least one antioxidant enzyme or polyalkylene glycol.
A multimeric protein, comprising at least one modified human haemoglobin protein as claimed in any preceding claim.
The multimeric protein according to claim 4, wherein the multimer is cross-linked.
A composition comprising:
a modified human haemoglobin protein as claimed in any one of claims 1 to 5; and

a pharmaceutically acceptable carrier or diluent.
The composition as claimed in claim 6, further comprising at least one reductant.
The composition according to claim 7, wherein the at least one reductant is ascorbate.
The composition as claimed in any one of claims 6 to 8, wherein the composition is a blood substitute composition.
The composition as claimed in any one of claims 6 to 9, for use as a medicament.
The composition as claimed in any one of claims 6 to 9, for use in the treatment of ischemia and/or hypoxia.